: Regulation of the actin cytoskeleton by cell signaling pathways is important for cellular movement and function. The activation of one Rho GTPase, Cdc42, leads to the rapid and transient polymerization of a highly-branched actin network nucleated by Arp2/3 complex. The event creates a myosin-independent force that pushes forward the membrane at the leading edge of motile cells. While much is known about the activation and deactivation of this process, important questions remain. How is formation of branched networks localized to the leading edge of the cell and not from actin filaments deeper in the cortex and in stress fibers? What protects selected populations of actin filaments from severing and pointed-end depolymerization by ADF/cofilin? Is there a hierarchy of filament stability? The goal of the research is to understand the cascade of events leading to the formation and maintenance of stable actin filaments in the cell. The research will explore the roles of ADF/cofilin, Arp2/3 complex, tropomyosin and tropomodulin, together with the actin nucleotide, in defining the pathway from nascent, branched actin filaments to stable filaments. There are four specific aims. 1) How are branched filaments converted to stable unbranched filaments and how do long, unbranched filaments remain unbranched?2) How are the pointed ends of stable actin filaments protected from depolymerization?3) How are long actin filaments in the deep cell cortex and stress fibers protected from severing and pointed end depolymerization by ADF/cofilin while those in the branched actin network are not?4) To determine the localization of tropomyosin isoforms in relation to Arp2/3 complex, cofilin, and regions of dynamic remodeling of the actin cytoskeleton. The methods for Aims 1-3 include measurement of actin polymerization and depolymerization using actin labeled with a fluorescent probe as well as microscopic analysis of fluorescently-labeled actin filaments. Aim 4 will localize the proteins as well as free filament ends in cells using indirect immunofluorescence. The results will help understand how the cytoskeleton is remodeled during complex functions such as leucocyte chemotaxis and growth factor-stimulated cellular outgrowth, processes important in both normal and pathological states.